Energy Transfer

1
Energy Transfer

• Chapter 13

2
Introduction

• Transfer of excited state energy from donor to
  acceptor
• Occurs without appearance of photon long range
  dipole-dipole interactions
• Depends on extent of overlap of emission spectrum
  of donor and absorption spectrum of acceptor, QY
  of donor, relative orientation of donor and
  acceptor transition dipoles, a distance between
  the donor and acceptor
• Applications
• distances between sites on a macromolecule
• Time resolved energy transfer

3
Introduction

• Occurs over distances comparable to dimensions of
  macromolecule
• Distance at which RET is 50 is called Forster
  distance (20-60 Å)
• When donor acceptor distance is equal to Forster
  distance transfer efficiency 50

RET depends strongly on distance (r6)
4
What can we use RET for?

• Distances between sites on a macromolecule
• How conformational changes affect those distances
• Conformational changes in multisubunit proteins
  association/dissociation
• Theory related to oscillating dipole
• Radiative energy transfer transfer emission and
  reabsorption inner filter effects
• NonRadiative energy transfer structural
  information
• Long range affects more structural information

5
Theory of Energy Transfer
kT(r) 1/?D(R0/r)6 E kT / ?D-1 kT ratio
of energy transfer rate to total decay rate If
the transfer rate is faster than the decay rate,
then efficiency will be high
6
Theory of Energy Transfer

• Measure the lifetime of donor in the absence and
  presence of the acceptor
• Steady-state measurement intensity in the
  presence and absence of acceptor. Can use
  decrease in donor or increase in acceptor to
  quantify.

7
Theory of Energy Transfer

• Strong dependence of energy transfer efficiency
  on R0.
• Most sensitive near R0
• Not useful away from R0

8
Overlap Integral

• J(?) - Overlap of excitation (acceptor) and
  emission(donor) spectra
• Comparison of overlap between different isomers
  of dansyl-DPE and eosin-labeled lipids.
• The R0 is not very sensitive to J(?) - sixth
  root dependence
• Visual impression not always the whole story
• Quantum yield of donor can change the R0

9
Orientation Factor

• ?T angle between the emission transition dipole
  of the donor and the absorption transition dipole
  of the acceptor
• ?D and ?A are the angles between these dipoles
  and the vector joining the donor and acceptor
• is the angle between the planes
• ?2 can range from 0 to 4
• ?2 4 , Colinear and parallel dipoles
• ?2 1, for parallel dipoles
• Variation in 1-4 only 26 change in r
• ?2 0, for perpendicular dipoles

?2 (cos?T 3cos?Dcos?A)2 (sin?Dsin?Acos?
2cos?Dcos?A)2
10
Experimentally Confirmed

• Many assumptions required testing
  experimentally
• Dependence on 1/r6 - Oligomers of poly-L-proline,
  labeled on opposite ends helix of known
  dimensions
• Dependence on overlap integral donor acceptor
  pair linked by a rigid steroid spacer extent of
  spectral overlap altered in different solvents
• No data to confirm the dependence of energy
  transfer on ?2
• Can also get homotransfer with fluorophores with
  small stokes shift

11
Distance Measurements in alpha-Helical Melittin
R0 23.6 Å E FDA/FD -1 0.45 r ?
12
?2 2/3 , good assumption because correlation
time of probes in ps range completely
randomized during the fluorescence
lifetime Fraction of acceptor labeling - can
dramatically affect measurements E
(1-FDA/FD)1/fA - acceptor labeling can effect
efficiency
13
Effect of ?2 on the Possible Range of Distance

• Set upper and lower limits for ?2 using
  anisotropy measurements
• Must know the depolarization due to segmental
  motions of probes
• Determine steady state (r) and fundamental
  anisotropy (r0)
• Fluorophores with mixed polarization r0lt 0.3, the
  error in distance is thought to be below 10
• Perin plot determine 1/r0app compare to frozen
  solution 1/r0 differences are due to segmental
  motions di (r0app/r0)1/2

14
Calmodulin MLCK Binding

• 1,5 IAEDANS to trp resides
• Quenching of trp fluorescence when IAEDANS Bound
• Degree of energy transfer was measured by
  comparing fluorescence of MLCK peptides free in
  solution to bound to labeled CaM

15
Association Kinetics of DNA

• One strand labeled with fluoroscein and the other
  labeled with rhodamine
• Binding of two complementary strands monitored by
  energy transfer
• Rates of association/dissociation
• Melting temperature
• Endonuclease digestion- elimation of RET by
  enzymatic cleavage of covalently linked D-A
• Many assays developed in this manner

16
Efficiency Enhanced Acceptor Emission

• Advantage if QY of donor changes it does not
  affect the efficiency
• Need to know the extinction coefficients of donor
  and acceptor at the excitation wavelength
• Need to know the fraction of the donor labeled

17
Myosin V 1IQ Labeled with FlAsH
Upper 50 kDa domain
FlAsH Site (residues 292-297)
ADPBeFx
FlAsH Structure
50 kDa Cleft
18
Steady-State Fluorescence
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FRET During Product Release
13.7 ? 0.5 s-1 195 ? 14 s-1
19.2 ? 1.5 s-1 200 ? 9 s-1
13.7 ? 0.3 s-1
MV FlAsH
MV (4-Cys) Unlabeled
MV FlAsH
1) Mant Fluorescence changes upon Pi-release 2)
FRET does not change upon Pi-release
mantATP
Actin
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FRET Sensors
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Rac Activation in Cells
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Energy Transfer in Solution

• Energy transfer also occurs with donors and
  acceptors randomly distributed in 3-D solution
• Concentration must be quite high to get
  significant energy transfer
• A0 acceptor concentration which results in 76
  energy transfer A0 447/R03
• If R0 25 Å, then A0 29 mM